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Editorial
Designing a molecular analysis of clonality in tumours
Salvador J. Diaz-Cano
Department of Histopathology and Morbid Anatomy, St Bartholomew's and the Royal London School of Medicine and Dentistry, Whitechapel, London
E1 1BB, UK
Abstract
Clonality analysis is used to test malignant transformation and tumour progression. X-chromosome
linked clonality assays have been employed for this purpose, but are subject to certain technical
limitations. This paper reviews the issues involved and the controls that are necessary to ensure
valid interpretation of such analyses.
Keywords: clonality; X-chromosome; lyonization; neoplasia
Clonality is an essential attribute of neoplasms and its
analysis has been used to test malignant transforma-
tion and tumour progression [1,2]. Concordant pat-
terns of genetic markers (X-linked or not) in different
tumours suggest that a common progenitor contribu-
ted to those lesions and favour, therefore, a multifocal
rather than a multicentric origin. These shared genetic
alterations also suggest a common cellular origin for
biphasic neoplasms [3]. Saxena et al. recently reported
a monoclonal pattern in smooth muscle cells and blood
vessels of sporadic angiomyolipoma, while the adipose
tissue revealed a polyclonal pattern [4]. Based on these
®ndings the authors concluded that the polyclonal
adipose tissue is probably metaplastic or reactive.
This represents a good example of the application of
clonality in tumour biology.
However, some biological and technical issues arise
from this article. X-linked clonality assays are based on
DNA polymorphism and random X-chromosome
inactivation (XCI) in females. Those features enable
us to distinguish the maternally from the paternally
inherited X-chromosomes [1,5,6]. The mechanisms
leading to XCI have not been fully characterized, but
DNA methylation might maintain the inactive state,
once it is established during early embryogenesis.
These methylation patterns are then transmitted by
clonal inheritance through the strong preference of
mammalian DNA (cytosine-5)-methyltransferase for
hemimethylated DNA, involving the promoter regions
of alleles on the inactive X-chromosome only [7]. Since
XCI analysis is based on differential DNA methylation
of one allele from X-chromosome genes (e.g. human
androgen receptor gene), suboptimal enzymatic diges-
tion and abnormal methylation can result in changes
of clonality patterns.
According to Lyon's hypothesis, all but one X-
chromosomes in a cell are randomly inactivated during
early embryogenesis, when the primordial cell pool
may comprise as few as 16±30 cells [8]. Given that
small number of embryo-destined cells, it reasonable to
expect unequal numbers of paternally- and maternally-
inherited inactive X-chromosomes, although the X-
chromosome is randomly inactivated in each cell. The
average Lyonization ratio is close to 50 : 50 in large cell
populations, although individual variation has been
found [8]. Skewing towards one allele to an extent that
meets the criteria for clonal derivation is consistent
with early XCI during embryogenesis (Figure 1).
This ®nding leads us to consider the selection of
appropriate controls to assess the Lyonization ratio in
each female. This ratio can also vary from tissue to
tissue in the same individual, due to unequal splitting
of the cells derived from the primordial cell pool, or to
different methylation patterns in different tissues [6,9].
Controls for unequal Lyonization should thus ideally
be the most closely related tissue thought not to be
involved in the disease process. An essential require-
Figure 1. Methylation pattern of androgen receptor alleles in
control samples. Only polymorphic and polyclonal controls (two
allele bands in both undigested and digested samples) are
considered informative for clonality assays (lanes 1 and 2). The
remaining possibilities (lanes 3±8) should be excluded from
clonality analyses, due to either monoclonal origin of controls
(lanes 3±6) or absence of locus polymorphism (lanes 7and 8).
U=undigested sample: D=digested sample
Journal of Pathology
J Pathol 2000; 191: 343±344.
Copyright #2000 John Wiley & Sons, Ltd.
ment for clonality analysis is the identi®cation of a
polymorphic locus in the normal control (Figure 1). In
every case, the tumour sample must be compared with
matched controls from the same patient to test the
heterozygosity for the marker. Additionally, the indi-
vidual variability and tissue-related Lyonization ratio
require samples of close embryological origin. This
feature must be maintained in the digested sample in
those tests based on XCI (Figure 1).
Positive allelic imbalances are determined case-by-
case, using the skewed data normalized by the allele
ratio in matched controls [1,6]. Allelic imbalance
analysis is based on the allele ratio and requires
densitometric analysis of both allele bands. Therefore,
the allele ratio in the target DNA must be maintained
in the ampli®cation product, which has to avoid the
PCR plateau phase. At this level, any PCR ampli®ca-
tion bias should be considered, especially DNA
degradation of the larger allele in formalin-®xed,
paraf®n-embedded tissues and defective ampli®cation
of repetitive CG-rich sequences [10±12].
Early XCI occurs randomly and results in a chess-
board pattern of cells descended from a common
progenitor, which may grow together like a clone
(patch size mosaicism). This pattern represents an
example of tissue heterogeneity that can also be present
in tumours. Sample size is a limiting factor; the lower
the cell number, the higher the probability of mono-
clonal patterns based on patch size mosaicism. This
concept becomes particularly important in mixed
tumours, where multiple microdissected samples from
different tumour areas (i100 cells) and from controls
are required to address the question.
Monoclonal patterns support a neoplastic rather
than a reactive or hyperplastic process, but are not
diagnostic of it. Host cell contamination of tumour
samples could give false heterozygous results that
would require careful microdissection and microscopic
control of the sample collection. However, the pitfalls
mentioned above should be always excluded.
Some of these considerations do not appear to have
been addressed in the paper of Saxena et al. [4],
especially those concerning tests for digestion comple-
tion with restriction endonuclease; controls regarding
both tumour heterogeneity and their methylation
patterns; PCR bias in the ampli®cation of both alleles;
tumour heterogeneity and patch size mosaicism; and
the meaning of monoclonal and polyclonal patterns.
References
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344 Editorial
Copyright #2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 343±344.